Retroviruses cause several diseases and pathological conditions, including a variety of tumors and leukemias. For instance, human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) in humans. Another significant disorder, adult T-cell leukemia-lymphoma, is caused by the retrovirus HTLV I (human t-cell leukemia virus type I). HTLV I also has been associated with other diseases, such as tropical spastic paraparesis and HTLV I-associated myelopathy. Moreover, many animal diseases of agricultural and veterinary importance are known to be caused by retroviruses. These include avian sarcoma leukosis virus (ASLV), feline leukemia virus (FeLV), bovine immunodeficiency virus (BIV) and equine infectious anemia virus (EIAV), among others.
Retroviruses encode several enzymes that are assembled into the virus particle and whose activities catalyze essential steps in the infectious cycle: (a) protease (PR), (b) the polymerase and (c) ribonuclease H(RNAse H) activities of reverse transcriptase (RT), and (d) integrase (IN). Following attachment and penetration of the retrovirus into the host cell, the RT activities catalyze reverse transcription of the viral RNA into DNA, and IN catalyzes the integration of the retroviral DNA into the host cell DNA. The cellular transcription and translation machinery is used to produce viral RNA from the integrated viral DNA and thereafter to produce the viral proteins.
Retroviral DNA integration proceeds in three distinct steps, the first two of which have been reconstituted in vitro with the purified retroviral enzyme, integrase. In the first step of integration, denoted processing, retroviral integrase removes two nucleotides from 3′-ends of the viral DNA. In the second step, joining, these newly created ends are joined to staggered phosphates in the host DNA in a concerted cleavage and ligation reaction. This process creates an integration intermediate that leaves gaps in the flanking host DNA sequence (FIG. 1). In the last step of integration, these gaps are repaired, creating a stably integrated provirus. This repair reaction can also be reconstituted in vitro using combinations of various polymerases, ligases, and an endonuclease (Brin et al., J. Biol. Chem. 275: 39287-39295, 2000; Yoder & Bushman, J. Virol. 74: 11191-11200, 2000), but the identity and mechanism of action of proteins responsible for these reactions in vivo are not yet known. Retroviral DNA integration is an essential step in retroviral replication and the integrase protein is an attractive target for antiviral therapy. However, the integrase gene is virus-encoded and therefore subject to a high mutation rate, leading to drug resistance. This rapid evolution of resistance should not occur with drugs that target cellular functions necessary for integration, but not cell viability.
As reported in WO 00/17386 and by Daniel et al. (Science 284: 644-647), retroviral infection induces programmed death in scid lymphocytes that are deficient in the DNA repair protein, DNA-PK (DNA-dependent protein kinase). Furthermore, this response to infection requires an active integrase. In addition to retrovirus-induced scid cell death, it was also observed that stable transduction by retroviral vectors, a measure of successful DNA integration, is reduced in cells deficient in DNA-PK and other components of the non-homologous end-joining (NHEJ) pathway. These findings indicate that retroviral DNA integration is sensed as DNA damage by the host cell, and that DNA repair proteins, such as DNA-PK, may be recruited to facilitate stable integration into the host genome. Components of the DNA damage response pathway(s) that are required for this process therefore present advantageous targets for anti-retroviral therapy.
DNA-PK belongs to a family of large, PI-3K-related protein kinases, that also includes ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3 related) kinases. The ATM and ATR kinases seem to have a broader role than DNA-PK in response to DNA damage, including regulation of cell cycle checkpoints. Detection of aberrant DNA and chromosome structures by these proteins coordinately triggers checkpoint pathways and DNA repair systems (Zhou & Elledge, Nature 408: 433-439, 2000). Activation of a DNA damage checkpoint results in cell cycle arrest, allowing time for DNA repair or, in its absence, cell death.
Cells derived from A-T patients and from atm −/− knockout mice display sensitivity to ionizing radiation, chromosomal instability, and defects in cell cycle checkpoints. The regulation of checkpoints by ATM has been studied extensively and a number of ATM substrates have been identified (Shiloh, Curr. Opin. Genet. Devel. 11: 71-77, 2001; Durocher & Jackson, Curr. Opin. Cell Biol. 13: 225-231, 2001). ATM also appears to play a direct role in DNA repair at the sites of DNA damage (Durocher & Jackson, 2001, supra). This ATM function may be mediated by modification of repair proteins, such as phosphorylation of BRCA1, which is induced by ionizing radiation (Cortez et al., Science 286: 1162-1166, 1999).
Studies with an inducible, transdominant-negative ATR mutant have also implicated the ATR protein in cell cycle checkpoint control (Cliby et al., EMBO J. 17: 159-169, 1998). Furthermore ATR, like ATM, can respond to DNA damage induced by ionizing radiation, and some data suggest that phosphorylation is sequential, with ATM kinase being activated first (Durocher & Jackson, 2001, supra). ATR and ATM seem therefore to be operating in similar or overlapping pathways (Shiloh et al., 2001, supra). However, the kinase activities of these proteins also have distinct functions—for example, BRCA1 is phosphorylated by ATR, but not ATM, in response to damage induced by UV light and stalled DNA replication forks (Tibbetts et al., Genes Dev. 14: 2989-3002, 2000).
In addition to phosphorylation of checkpoint and DNA repair proteins, ATM and ATR also share in vitro and in vivo sensitivity to the radiosensitizing agent caffeine (Zhou et al., J. Biol. Chem. 275: 10342-10348, 2000; Blasina et al., Curr. Biol. 9: 1135-1138, 1999; Hall-Jackson et al., Oncogene 18: 6707-6713, 1999). The IC50s for ATM and ATR kinase inhibition are similar, and fall in the range of 1-2 mM in vitro (Sarkaria et al., Cancer Res. 59: 4375-4382, 1999). Although ATM function is required for the residual retroviral transduction that occurs in cells that are deficient in NHEJ proteins such as DNA-PK, retroviral transduction is normal in ATM-deficient cells (WO 00/17386; Daniel et al., Mol. Cell. Biol. 21: 1164-1172, 2001). Further, DNA-PK is not sensitive to caffeine (Sarkaria et al., 1999, supra).